The invention relates to a retro-inverso gonadotropin-releasing hormone (RI-GnRH) peptide and the use thereof as a vaccine.
The decapeptide gonadotropin releasing hormone (GnRH) regulates the reproductive hormone cascade by stimulating the release of gonadotropins from the anterior pituitary, which in turn regulates reproductive function. GnRH is synthesised in the neurones of the hypothalamus and released into the portal circulation where it interacts with GnRH receptors on the gonadotrope cells in the anterior pituitary [6]. Stimulation of the GnRH receptor is essential for the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which in turn are required for steroidogenesis and gametogenesis, respectively [6]. Due to this central role in reproduction, GnRH peptide analogs have found therapeutic applications in controlling fertility, cryptorchidism, polycystic ovarian syndrome, leiomyomata, endometriosis, acute intermittent porphyria, and breast, ovarian and prostatic cancer [3, 16].
It has been reported that up to 80% of female cattle sent to abattoirs are pregnant. As some of the feed ingested by pregnant cattle is diverted to the unborn calves, especially during the last three months of gestation, there is a loss of body tissue in adult female cattle and thus less beef is obtained from these cattle. This results in a loss of value of the pregnant animal.
Recognition of the importance of avoiding pregnancy in female cattle which are destined for slaughter has led to various methods to avoid fertility, such as surgical spaying. This is an unpleasant procedure with several side effects, and additionally, permanent sterilisation is not always desirable. For example, in the beef and dairy industry, pregnancy too soon after calving is a major problem, and it would be desirable to induce a temporary state of infertility in these cattle to allow them to recover after calving.
There is also a need for controlling gonadal activity in other domestic animals and wild animals, and in chronic diseases in humans.
Peptide-based vaccines which can be administered to cattle by way of injection to reduce their fertility are known [1, 17]. These vaccines against L-amino acid native GnRH conjugates stimulate the animals' immune systems to produce antibodies that block the hormonal pathway involved in reproduction. The vaccines have subsequently also undergone clinical trials in treatment of prostate cancer in humans [22] and have potential in sex hormone-dependent male and female cancers [8, 21]. Antibodies to GnRH have also been raised in a number of species by chemical conjugation of GnRH to a suitable carrier and administration of the conjugate in an appropriate adjuvant. Recombinant fusion proteins comprising GnRH or GnRH-analogues have also been described for use in peptide vaccines for the immunological castration or inhibition of reproductive function of various domesticated and farm animals.
The advantages of peptide based vaccines are well described as they are chemically defined, are indefinitely stable and can be stored as a freeze dried powder. The preparation does not require large-scale production and is relatively cheap. However, a major limitation of peptide vaccines is their relatively low immunogenicity and limited biological half-life [20]. In this regard, prior GnRH constructs have failed to provide a uniformly successful immunological sterilization vaccine product due to the fact that GnRH is a small “self” molecule that is not normally recognized by a subject's immune system, rendering the molecule poorly immunogenic and inherently unable to induce a significant immune response against endogenous GnRH. Chemical conjugation protocols are also difficult to control, rendering substantially heterogenous and poorly-defined GnRH conjugates. The peptide nature of GnRH vaccines has necessitated administration by means of injection along with adjuvant.
An alternative approach to immunization with native peptides is the use of peptidomimetics such as retro-inverso (RI) peptides that could serve as vaccines. RI peptides which produce antibodies directed against large polypeptides of foot and mouth disease virus are known. Antibodies to these RI peptides have been reported to show greater affinity than antibodies to classical L peptides and show strong neutralizing activity. However, RI peptides which are directed to small biologically active peptides such as GnRH have not been reported. As the N and C termini (pGlu and Gly-NH2), which are important for binding of GnRH to its cognate receptor, cannot be simulated in RI-GnRH [NH2—CH(C2H4COOH)—CO— and NH2—CH—CO—], and because of the many differences between small peptides and large polypeptides, it is not predictable that antibodies raised against RI-GnRH would immunoneutralise the native peptide.
Thus, although a variety of contraceptive methods are available to control fertility, each has disadvantages such as affordability, application difficulty (injections), daily intake (pills) and irreversible procedures (surgical methods).
There is therefore a demand for an improved and a cost-effective approach to regulate reproductive function and sex hormone-related diseases. The development of a potent immunogenic active GnRH vaccine would therefore greatly enhance the utility of this pharmaceutic agent in current therapies.